Effect of H1N1 Swine Virus on Humans

Effect of H1N1 Swine Virus on Humans How does the new H1N1 swine virus infect humans compared to the common influenza virus? SUMMARY Pandemic influenza viruses cause significant mortality in humans. In the 20th century, there are 3 influenza viruses which caused major pandemics: the 1918 H1N1 virus, the 1957 H2N2 virus, and the 1968 H3N2 virus. All three aforementioned pandemics were caused by viruses containing human adapted PB2 genes. In March and early April 2009, a new swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the United States. During the first few weeks of strain surveillance, the virus spread worldwide to many countries by human-to-human transmission (and perhaps due to the airline travel). In 2 months time, 33 countries had officially reported 5.728 cases resulting in 61 deaths, and by June 2009 WHO reported 30 000 confirmed cases in 74 countries. On June 11 of 2009, this led the World Health Organization (WHO) to raise its pandemic alert to level 5 (Human-to-human spread of the virus into at least 2 countries in 1 WHO region) of 6 (Human-to-human spread of the virus into at least 1 other country in a different WHO region in addition to phase 5 criteria). According to the sayings of Smith et al. (2009), this virus had the potential to develop into the first influenza pandemic of the twenty-first century. In the early summer of 2009, the causes of the human infection and influenza spread among humans had still remained unknown although many publications of that period tried to elucidate this influenza outburst. For example, according to the sayings of Palese, the new H1N1 could also die out entirely. â€Å"Theres a 50-50 chance it will continue to circulate†, he predicts. Conclusively, in that early period, the fuzziness of the data about this new viruss behaviour led scientists only to speculate using past data. Today the 2009 H1N1 virus has ultimately created the first influenza pandemic, has disproportionately affected the younger populations (which perhaps reflects the protection in the elderly due to their exposure to H1N1 strains before 1957), bu t turned out to be not highly pathogenic because the majority of cases of 2009 influenza A H1N1 are mild. Genomic analysis of the 2009 influenza A (H1N1) virus in humans indicates that it is closely related to common reassortant swine influenza A viruses isolated in North America, Europe, and Asia. Therefore, it contains a combination of swine, avian, and human influenza virus genes. More studies need be conducted to identify the unrecognized molecular markers for the ability of S-OIV A (2009 H1N1) to replicate and be transmitted in humans. As a result these additional studies would help us to determine the mechanism by which an animal influenza A virus crossed the species barrier to infect humans. Additionally, these molecular determinants can be used to predict viral virulence and pathogenicity for diagnosis. 1. LITERATURE REVIEW 1.1. Introduction â€Å"Swine flu† †influenza A [Family Orthomyxoviridae (like influenza B and C viruses), Genus Influenzavirus A] is currently the greatest pandemic disease threat to humankind (Salomon and Webster, 2009). The incidence and spread in humans of the â€Å"swine flu† influenza A virus has raised global concerns regarding its virulence and initially regarding its pandemic potential. The main cause of the â€Å"swine flu† has been identified to be the human infection by influenza A viruses of a new H1N1 (hemagglutinin 1, neuraminidase 1) subtype, or â€Å"2009 H1N1 strain† (Soundararajan et al., 2009) that contains genes closely related to swine influenza (SI) [also called swine flu, hog flu and pig flu]. Thus, the strains of virus that cause the annual seasonal flu are different than the new swine flu viruses that emerged in the spring of 2009. Consequently, as it will be analyzed in the subsequent chapters, the new swine flu virus has a unique combinatio n of gene segments from many different sources (a combination that has not been previously reported among swine or human influenza viruses) and specifically is thought to be a mutation of four known strains of the influenza A virus, subtype H1N1: 1. one endemic in (normally infecting) humans, 2. one endemic in birds, 3. and two endemic in pigs (swine). According to Yoon and Janke (2002), the constant evolution of influenza A viruses through mutation and reassortment present a complex and dynamic picture which is to be unfolded in the remaining Literature Review section more specifically for the H1N1 2009 virus. 1.2. Influenza Influenza is historically an ancient disease of global dimension that causes annual epidemics and, at irregular intervals, pandemics. Influenza is an infection of the respiratory tract caused by the influenza virus (see  § 1.3). When compared with the majority of other viral respiratory infections (such as the common cold), the infection by influenza often causes a more severe illness (Smith, 2003). Influenza-like illness (ILI) is defined by the CDC (Centers for Disease Control and Prevention) as fever (with temperature above 37,8 °C) and either cough or some throat in the absence of any other known cause. According to Webster (1999), influenza is the paradigm of a viral disease in which the continued evolution of the virus is of paramount importance for annual epidemics and occasional pandemics of disease in humans which is attributed to the fact that the H1N1 virus does not fit to the strict definition of a new subtype for which most of the population has not any experience of previous infection (Sullivan et al, 2010) as it is justified later in this Literatute Review section ( § 1.8). Influenza is transmitted by inhalation of microdroplets (because the transmission via large-particle droplets requires close contact which is attributed to the fact that these large-particle droplets cannot remain suspended in the air for a long period of time) of respiratory secretions, often expelled by coughing or sneezing, that contain the virus or from other bodily fluids (such as fomites, diarrheal stool etc.). The incubation period is between 1 to 5 days. Symptoms typically include fever, headache, malaise, myalgia, cough, nasal discharge, and sore throat. In severe cases of influenza, a secondary bacterial pneumonia can lead to the death of a patient (Suguitan and Subbarao, 2007). Vaccination and antiviral treatment constitute the two major options for controlling influenza and are the most effective means of preventing influenza virus infection and further transmission in humans. 1.2.1. Pandemic Influenza An influenza pandemic is a large-scale global outbreak of the disease, whereas an epidemic is considered more sporadic and localized. The aforementioned (in the Summary section) situation of pandemic influenza occurs when a previously circulated human influenza A virus [although all the three types (A, B, and C) of influenza viruses can infect humans)] acquires novel antigenic determinants from an animal-origin influenza virus and now can infect and propagate in humans in the absence of any pre-existing immunity (see  § 1.7 for details). Several influenza subtypes have infected humans. Historical accounts led us to consider that an average of three influenza pandemics have occurred each century, at intervals ranging from 10 to 50 years (Garcia-Sastre, 2005). The three influenza pandemics which occurred in the previous (20th) century are: 1. The â€Å"Spanish† influenza pandemic of 1918 (H1N1 subtype), 2. The 1957 â€Å"Asian flu† (H2N2), and 3. The 1968 ‘‘Hong Kong flu (H3N2). These pandemics resulted in high morbidity, death, and also considerable social and economic disruption. They provide health authorities information on which to base preparations for a future pandemic.The first influenza pandemic of the 21st century, due to a new strain of A(H1N1) virus, was declared on 11 June 2009 by the Director-General of the World Health Organization (WHO) [Collin et al., 2009] by raising the H1N1 flu virus pandemic alert level to phase 6 as it was mentioned in the Summary section. Although influenza B viruses do not cause pandemics, during some epidemic years they have caused more significant mortality and morbidity than influenza A viruses (FLUAV) [Garcia-Sastre, 2005]. 1.3. Influenza Virus It was already mentioned that influenza viruses are divided into three types designated A, B, and C (according to the antigenic differences of their internal structural components as it is discussed below in the current chapter). Influenza types A and B are responsible for epidemics of respiratory illness that occur almost every winter and are often associated with increased rates for hospitalization and death. As it was mentioned in the previous chapter, influenza A virus has also the capability of developing into pandemic virus. Type C infection usually causes either a sporadic mild or asymptomatic respiratory illness or no symptoms at all (Smith, 2003). In comparison to B and C influenza types which are specific to humans, type A viruses can have different hosts, both birds and different mammals (e.g. horses and pigs) including humans (Ã…sjà ¶a and Kruse, 2007). Specifically, influenza B virus strains appear to infect naturally only humans and have caused epidemics every few years (Schmitt and Lamb, 2005). On the other hand, influenza A viruses are significant animal pathogens of poultry, horses and pigs, and multiple antigenically diverse strains exist in a aquatic wild bird reservoir (Garcia-Sastre, 2005). Migrating aquatic birds carry viruses between the continents and thereby play a key role in the continuing process of virus evolution (Murphy et al., 1999). Influenza C virus causes more limited outbreaks in humans and according to Schmitt and Lamb (2005) also infects pigs. Although influenza viruses belong to the best studied viruses, according to Haller et al. (2008), the molecular determinants, which govern the increased virulence of emerging virus strains in humans and which may be associated with their transmission and transmissibility, are presently not well understood. Influenza viruses are negative-strand RNA[1] viruses with a segmented genome (which replicates in the nucleus of the infected cell) belonging to the Orthomyxoviridae family. The morphology of the influenza virion is described in the next chapter. On the basis of antigenic differences influenza viruses are divided into influenza virus types A, B and C. Influenza A viruses are classified on the basis of the antigenic properties of their haemagglutinin (H or HA) and their neuraminidase (N or NA) structural spike-shaped surface glycoproteins (antigens): to date, 16HA (H1-H16) and 9NA (N1-N9) subtypes have been identified (Osterhaus et al., 2008) which gives a theoretical possibility of 144 serological subtypes. Subtypes of influenza A viruses are constantly undergoing small antigenic modifications (antigenic drift) [which is a serotypic change] due to the accumulation of point mutations in their genetic material. In addition, due to the segmented genome, genetic reassortment occurs perio dically when HA and NA genetic material is exchanged between viruses, thereby causing major antigenic changes (antigenic shift) [Yoon and Janke, 2002], the emergence of a new subtype (Smith, 2003) and perhaps the potential for a pandemic outbreak. Both antigenic shift and drift are discussed in  § 1.7. The family Orthomyxoviridae, except the aforementioned influenza viruses A, B and C, also contains the Thogoto viruses. Thogoto viruses are transmitted by ticks and replicate in both ticks and in mammalian species and are not discussed as part of this assignment (Schmitt and Lamb, 2005). 1.4. Influenza Virus Virion This paragraph describes the (belonging to the Orthomyxoviridae family) virus virion[2] morphology. These virions are spherical or pleomorphic, 80-120 nm in diameter (see 1). Some of them have filamentous forms of several micrometers in length. The virion envelope[3] is derived from cell membrane lipids, incorporating variable numbers of virus glycoproteins (1-3) and nonglycosylated proteins (1-2) [Fauquet et al., 2005]. 1. (Left) Diagram of an Influenza A virus (FLUAV) virion in section. The indicated glycoproteins embedded in the lipid membrane are the trimeric hemagglutinin (HA), which predominates, and the tetrameric neuraminidase (NA). The envelope also contains a small number of M2 membrane ion channel proteins. The internal components are the M1 membrane (matrix) protein and the viral ribonucleoprotein (RNP) consisting of RNA segments, associated nucleocapsid protein (NP), and the PA, PB1 and PB2 polymerase proteins. NS2 (NEP), also a virion protein, is not shown (Fauquet et al., 2005). (Right) Negative contrast electron micrograph of particles of FLUAV. The bar represents 100 nm (Fauquet et al., 2005). The lipid envelope is derived from the plasma membrane of the cell in which the virus replicates and is acquired by a budding process (see  § 1.5) from the cell plasma membrane as one of the last steps of virus assembly and growth (Schmitt and Lamb, 2005) which is initiated by an interaction of the viral proteins. Virion surface glycoprotein projections are 10-14 nm in length and 4-6 nm in diameter. The viral nucleocapsid (NP) is segmented, has helical symmetry, and consists of different size classes, 50-150 nm in length (Fauquet et al., 2005). The nucleocapsid segments (the number of which depends on the virus type) surround the virion envelope which has large glycoprotein peplomers (HA, NA, HE). There are two kinds of glycoprotein peplomers[4]: (1) homotrimers of the hemagglutinin protein (NA) and (2) homotetramers of the neuraminidase protein (NA) [see 1 and 2]. Influenza C viruses have only one type of glycoprotein peplomer, consisting of multifunctional hemagglutinin-esterase molecules (HE) [see  § 1.4.1 for further details]. Genomic segments have a loop at one end and consist of a molecule of viral RNA enclosed within a capsid composed of helically arranged nucleoprotein (NP) as it is shown in 2 (Murphy et al., 1999). 2. Schematic representation of an influenza A virion showing the envelope in which three different types of transmembrane proteins are anchored: the hemagglutinin (HA) and the neuraminidase (NA) form the characteristic peplomers and the M2 protein, which is short and not visible by electron microscopy. Inside the envelope there is a layer of M1 protein that surrounds eight ribonucleoprotein (RNP) structures, each of which consists of one RNA segment covered with nucleoprotein (NP) and associated with the three polymerase (P) proteins (Murphy et al., 1999). The aforementioned in the previous paragraph NP protein (arginine-rich protein of approximately 500 amino acids) is the major structural protein of the eight RNPs and it has been found to be associated with the viral RNA segments. Each NP molecule covers approximately 20 nucleotides of the viral RNAs. The NP mediates the transport of the incoming viral RNPs from the cytoplasm into the nucleus by interacting with the cellular karyopherin/importin transport machinery. In addition, the NP plays an important role during viral RNA synthesis, and free NP molecules are required for full-length viral RNA synthesis, but not for viral mRNA transcription (Palese and Garcia-Sastre, 1998). 1.4.1. Influenza Viral Proteins Influenza A and B viruses possess eight single-stranded negative-sense RNA segments (see 2) that encode structural and nonstructural proteins [NS][5]: 1. Hemagglutinin (HA), a structural surface glycoprotein that mediates viral entry (see  § 1.5 for further details) by binding (the HA1 fragment) to sialic acid residues (present on the cell surface) on host fresh target cells, is the main target of the protective humoral immunity responses in the human host (Suguitan and Subbarao, 2007). HA is primarily responsible for the host range of influenza virus and immunity response of hosts to the infection (Consortium for Influenza Study at Shanghai, 2009). After the binding, the virus is taken up into the cell by endocytosis. At this point, the virus is still separated by the endosomal membrane from the replication and translation machinery of the cell cytoplasm (Fass, 2003). HA is initially synthesized and core-glycosylated in the endoplasmic reticulum (ER)[6] as a 75-79 kDa precursor (HA0) which assembles into noncovalently linked homo-trimers. The trimers are rapidly transported to the Golgi complex and reach the plasma membrane, whe re HA insertion initiates the process of assembly and maturation of the newly formed viral particles (33-35). Just prior to or coincident with insertion into the plasma membrane, each trimer subunit is proteolytically and posttranslationally cleaved into two glycoproteins (polypeptides), HA1 and HA2 ( 3), which remain linked by a disulfide bond (Rossignol et al., 2009) and associated with one another to constitute the mature HA spike (a trimer of heterodimers). In that way, the membrane fusion during infection is promoted. Cleavage activates the hemagglutinin (HA), making it ready to attach to receptors on target cells (Murphy et al., 1999). Conclusively and in addition, the HA undergoes various post-translational modifications during its transport to the plasma membrane, including trimerization, glycosylation, disulfide bond formation, palmitoylation, proteolytic cleavage and conformational changes (Palese and Garcia-Sastre, 1998). HA1 is the subunit distal from the virus envelope, whereas HA2 contains a hydrophobic region near the carboxy terminus that anchors the HA1-HA2 complex in the membrane ( 3) [Fass, 2003]. The HA complex is brought to the cell surface via the secretory pathway and incorporated into virions, along with a section of cell membrane, as the virus buds from the cell. HA1 is the subunit distal from the virus envelope, whereas HA2 contains a hydrophobic region near the carboxy terminus that anchors the HA1-HA2 complex in the membrane (see 3) [Fass, 2003]. 3. Primary structure of influenza HA and spatial organization of subunits with respect to the membrane. Cleavage of the influenza HA precursor protein HA0 yields the two subunits HA1 and HA2. HA1 is white, the fusion peptide and transmembrane segments of HA2 are black, and the remainder of HA2 is cross-hatched. For clarity, a monomer of the HA1-HA2 assembly is shown. The amino and carboxy termini of HA2 are labelled ‘‘N and ‘‘C, respectively (Fass, 2003). 2. Neuraminidase (NA) is the other major surface glycoprotein, whose enzymatic function allows the release of newly formed virions, permits the spread of infectious virus from cell to cell, and keeps newly budding virions from aggregating at the host cell surface. This catalytic function of the NA protein is the target of the anti-influenza virus drugs oseltamivir (Tamiflu[7]) and zanamivir (Relenza7). Although these compounds do not directly prevent the infection of healthy cells, they limit the release of infectious progeny viruses thus inhibiting their spread and shortening the duration of the illness. These NA inhibitors are effective against all NA subtypes among the influenza A viruses and may be the primary antiviral drugs in the event of a future pandemic as it proved true in the current â€Å"swine flu† influenza A outbreak. Antibodies to the NA protein do not neutralize infectivity but are protective (Suguitan and Subbarao, 2007). Influenza C viruses lack an NA protein, and all attachment, entry and receptor destroying activities are performed by the aforementioned single spike glycoprotein: hemagglutinin-esterase-fusion (HEF) protein (Garcia-Sastre, 2005). The HEF protein distinguishes the antigenic variants of the genus C of the Orthomyxoviridae family, and the antibody to HEF protein neutralizes infectivity (Schmitt and Lamb, 2005). Of the three virus types, A and B viruses are much more similar to each other in genome organization and protein homology than to C viruses, which suggests that influenza C virus diverged well before the split between A and B viruses (Webster, 1999). Three proteins comprise the viral polymerase of the influenza viruses: two basic proteins (PB1 and PB2) and an acidic protein (PA). They are present at 30 to 60 copies per virion. The RDRP (RNA-dependent RNA polymerase) complex consists of these 3 polymerase proteins (Lamb and Krug, 2001). Together with the aforementioned scaffold protein NP (helically arranged nucleoprotein), these three polymerase proteins associate with the RNA segments to form ribonucleoprotein (RNP) complexes (Murphy et al., 1999). Thus, the RNPs contain four proteins and RNA. Each subunit of NP associates with approximately 20 bases of RNA (Lamb and Krug, 2001). The RNP strands usually exhibit loops at one end and a periodicity of alternating major and minor grooves, suggesting that the structure is formed by a strand that is folded back on itself and then coiled on itself to form a type of twin-stranded helix (Schmitt and Lamb, 2005). RDRP transcribes the genome RNA segments into messenger RNAs (mRNA). The RDR P complex carries out a complex series of reactions including cap binding, endonucleolytic cleavage, RNA synthesis, and polyadenylation[8]. The PA protein may be involved in viral RNA replication and, in addition, the expression of the PA protein in infected cells has been associated with proteolytic activity. The functional significance of the latter activity is not yet understood (Palese and Garcia-Sastre, 1998). Two viral RNA segments (7 and 8) encode at least two proteins each by alternative splicing. Gene segment 7 (see 4) codes for two proteins: matrix protein M1, which is involved in maintaining the structural integrity of the virion, and M2, an integral membrane (surface) protein that acts as an ion channel and facilitates virus uncoating. It is widely believed that the M1 protein interacts with the cytoplasmic tails of the HA, NA, and M2 (or BM2) proteins and also interacts with the ribonucleoprotein (RNP) structures, thereby organizing the process of virus assembly (Schmitt and Lamb, 2005). The drugs amantadine and rimantadine bind to the influenza A M2 protein and interfere with its ability to transport hydrogen ions into the virion, preventing virus uncoating. Amantadine is only effective against influenza A viruses (Suguitsan and Subbarao, 2007). Therefore, for the antiviral therapy, there are two classes of drugs which are currently available for the chemoprophylaxis and the treatment of influenza (Rossignol et al., 2009). These include the aforementioned NA inhibitors oseltamivir and zanamivir, which impair the efficient release of viruses from the infected host cell, and amantadine and rimantadine, which target the viral M2 protein required for virus uncoating. Passively transferred antibodies to M2 can protect animals against influenza viruses, but such M2-specific antibodies are not consistently detected in human convalescent sera (Black et al., 1993), suggesting that this type of immunity may play a minor role in the clearance of influenza virus in humans. Gene segment 8 (see 4) is responsible for the synthesis of the nonstructural protein NS1 and nuclear export protein (NEP, formerly called NS2) [Murphy et al., 1999] which is a minor structural component of the viral core and that mediates nucleo-cytoplasmic trafficking of the viral genome (Garcia-Sastre, 2005). NEP (NS2) plays a role in the export of RNP from the nucleus to the cytoplasm. NS1 protein suppresses the antiviral mechanism in host cells upon viral infection (Chang et al., 2009) and is involved in modulating the hosts interferon response (Garcia-Sastre, 2005). Recently, an unusual 87-amino acid peptide arising from an alternative reading frame of the PB1 RNA segment has been described (Chen et al., 2001). This protein, PB1-F2, is believed to function in the induction of apoptosis[9] as a means of down-regulating the host immune response to influenza infection. Specifically, it appears to kill host immune cells following influenza virus infection. It has been called the influenza death protein (Chen et al., 2001). PB1 segment encodes this second protein from the +1 reading frame. This protein consists of 87-90 amino acids (depending on the virus strain). This protein is absent in some animal, particularly swine, virus isolates. PB1-F2 protein is not present in all human influenza viruses. Human H1N1 viruses encode a truncated version. However, it is consistently present in viruses known to be of increased virulence in humans, including the viruses that caused the 1918, 1957, and 1968 pandemics. PB1-F2 localizes to mitochondria and treatment of cells with a synthetic PB1-F2 peptide induces apoptosis9 (Neumann et al., 2008). 4. Orthomyxovirus genome organization. The genomic organization and ORFs are shown for genes that encode multiple proteins. Segments encoding the polymerase, hemagglutinin, and nucleoprotein genes are not depicted as each encodes a single protein. (A) Influenza A virus segment 8 showing NS1 and NS2 (NEP) mRNAs and their coding regions. NS1 and NS2 (NEP) share 10 amino-terminal residues, including the initiating methionine. The open reading frame (ORF)[10] of NS2 (NEP) mRNA (nt 529-861) differs from that of NS1. (B) Influenza A virus segment 7 showing M1 and M2 mRNAs and their coding regions. M1 and M2 share 9 amino-terminal residues, including the initiating methionine; however, the ORF of M2 mRNA (nt 740-1004) differs from that of M1. A peptide that could be translated from mRNA has not been found in vivo. (C) Influenza A virus PB1 segment ORFs10. Initiation of PB1 translation is thought to be relatively inefficient based on Kozaks rule[11], likely allowing initiation of PB1-F2 translation by ribosomal scanning (Fauquet et al., 2005). In the same way, the M2 protein is anchored in the viral envelope of the influenza A virus, the ion channel proteins BM2 (it is encoded by a second open reading frame10 of RNA segment 7 of influenza B virus, and its function has not been determined) and CM2 are contained in influenza B and C viruses respectively ( 5). The CM2 protein is most likely generated by cleavage of the precursor protein. The influenza B viruses encode one more transmembrane protein, or NB, of unknown function (Garcia-Sastre, 2005). The cellular receptor for the influenza C virus is known to be the 9-0-acetyl-N-acetylneuraminic acid, and its receptor-destroying enzyme is not an NA, as it was already mentioned, but a neuraminate-O-acetylesterase. Like the HA protein of A and B viruses, the HEF of influenza C viruses must be cleaved in order to exhibit membrane fusion activity (Palese and Garcia-Sastre, 1998). 1.5. Viral Entry Influenza virus infection is spread from cell to cell and from host to host in the form of infectious particles that are assembled and released from infected cells. A series of events must occur for the production of an infectious influenza virus particle, including the organization and concentration of viral proteins at selected sites on the cell plasma membrane, recruitment of a full complement of eight RNP segments to the assembly sites, and the budding and release of particles by membrane fission (Schmitt and Lamb, 2005). Viral entry is a multistep process that follows at ­tachment of the virion to the cellular receptor and re ­sults in deposition of the viral genome (nucleocapsid) in the cytosol[12] (receptor-mediated endocytosis). The entry of enveloped viruses is exemplified by the influenza virus ( 6). The sequential steps in entry include (Nathanson, 2002):  § Attachment of the HA spike [the virus attachment protein (VAP)] to sialic acid receptors (bound to glycoproteins or glycolipids) on the cellu ­lar surface (see  § 1.4.1 for further details). This step contributes to pathogenesis, transmission, and host range restriction.  § Internalization of the virion into an endocytic vacuole.  § Fusion of the endocytic vacuole with a lysosome[13], with marked lowering of the pH (see 6). In endosomes, the low pH-dependent fusion occurs between viral and cell membranes. For influenza viruses, fusion (and infectivity) depends on the cleaved virion HA (FLUAV and FLUBV: HA1, HA2; FLUCV: HEF1, HEF2) [Murphy et al, 1999]. The infectivity and fusion activity are acquired by the post-translational cleavage of the HA of the influenza viruses which is accomplished by cellular proteases. Cleavability depends, among other factors, on the number of basic amino acids at the cleavage site. It produces a hydrophobic amino terminal HA2 molecule (Fauquet et al., 2005). 6. Diagram of the stepwise entry of influenza virus at a cellular level. Key events are attachment of the virion; internalization of the virion by endocytosis; lowering the pH of the endocytic vacuole leading to drastic reconfiguration of the viral attachment protein (hemagglutinin, HA1 and HA2); insertion of a hydrophobic domain of HA2 into the vacuolar membrane; fusion of the viral and vacuolar membranes; release of the viral nu ­cleocapsid into the cytosol (Nathanson, 2002).  § A drastic alteration in the structure of the HA1 trimer, with reorientation of the HA2 peptide to insert its proximal hydrophobic domain into the vacuolar membrane (Nathanson, 2002).  § Fusion of viral and vacuolar membranes (Nathanson, 2002).  § Integral membrane proteins migrate through the Golgi apparatus to localized regions of the plasma membrane (Fauquet et al., 2005).  § New virions form by budding, thereby incorporating matrix protein and the viral nucleocapsids which align below regions of the plasma membrane containing viral envelope proteins. Budding is from the apical surface in polarized cells (Fauquet et al., 2005).  § Release of the viral nucleocapsid into the cy ­tosol: After the formation of fusion pores, viral ribonucleoprotein complexes (RNPs) are delivered into the cytosol. RNPs are then transported into the nucleus, where transcription and replication occurs (see 7) [Garten and Klenk, 2008]. How the replication and the transcription of the genome of influenza virus take place in the nuclei of infected cells is summarized in detail by Palese and Garcia-Sastre (1998) [ 7]. (1) Adsorption: the virus interacts with sialic acid-containing cell receptors via its HA protein, and is intenalized by endosomes. (2) Fusion and uncoating: the HA undergoes a conformational change mediated by the acid environment of the endosome, which leads to the fusion of viral and cellular membranes. The inside of the virus also gets acidified due to proton trafficking through the M2 Ion channel. This acidification is responsible for the separation of the M1 protein from the ribonucleoproteins (RNPs), which are then transported into the nucleus of the host cell thanks to a nuclear localization Signal in the NP. (3) Transcription and replication: the viral RNA (vRNA) is transcribed and replicated in the nucleus by the viral polymerase. Two different species of RNA are synthesized from the vRNA template: (a) full-length copies (cRNA), which are used by the polymerase to produce more vRNA molecules; and (b) mRNA. (4) Translation: following export into the cytoplasm the mRNAs are translated to form viral proteins. The membrane proteins (HA, NA and M2) are transported via the rough endoplasmic reticulum (ER) and Golgi apparatus to the plasma membrane. The viral proteins possessing nuclear signals (PB1, PB2, PA, NP, M1, NS1 and NEP) are transported into the nucleus. (5) Packaging and budding: the newly synthesized NEP protein appears to facilitate the transport of the RNPs from the nucleus into the cytoplasm by bridging the RNPs with the nuclear export machinery. M1-RNP complexes are formed which interact with viral proteins in the plasma membrane. Newly made viruses bud from the host cell membrane (Palese and Garcia-Sastre, 1998). 1.5.1. Sialic Acid Receptors of Influenza Viruses Sialic acids (Sias) are a family of negatively charged 9-carbon sugars typically occ Effect of H1N1 Swine Virus on Humans Effect of H1N1 Swine Virus on Humans How does the new H1N1 swine virus infect humans compared to the common influenza virus? SUMMARY Pandemic influenza viruses cause significant mortality in humans. In the 20th century, there are 3 influenza viruses which caused major pandemics: the 1918 H1N1 virus, the 1957 H2N2 virus, and the 1968 H3N2 virus. All three aforementioned pandemics were caused by viruses containing human adapted PB2 genes. In March and early April 2009, a new swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the United States. During the first few weeks of strain surveillance, the virus spread worldwide to many countries by human-to-human transmission (and perhaps due to the airline travel). In 2 months time, 33 countries had officially reported 5.728 cases resulting in 61 deaths, and by June 2009 WHO reported 30 000 confirmed cases in 74 countries. On June 11 of 2009, this led the World Health Organization (WHO) to raise its pandemic alert to level 5 (Human-to-human spread of the virus into at least 2 countries in 1 WHO region) of 6 (Human-to-human spread of the virus into at least 1 other country in a different WHO region in addition to phase 5 criteria). According to the sayings of Smith et al. (2009), this virus had the potential to develop into the first influenza pandemic of the twenty-first century. In the early summer of 2009, the causes of the human infection and influenza spread among humans had still remained unknown although many publications of that period tried to elucidate this influenza outburst. For example, according to the sayings of Palese, the new H1N1 could also die out entirely. â€Å"Theres a 50-50 chance it will continue to circulate†, he predicts. Conclusively, in that early period, the fuzziness of the data about this new viruss behaviour led scientists only to speculate using past data. Today the 2009 H1N1 virus has ultimately created the first influenza pandemic, has disproportionately affected the younger populations (which perhaps reflects the protection in the elderly due to their exposure to H1N1 strains before 1957), bu t turned out to be not highly pathogenic because the majority of cases of 2009 influenza A H1N1 are mild. Genomic analysis of the 2009 influenza A (H1N1) virus in humans indicates that it is closely related to common reassortant swine influenza A viruses isolated in North America, Europe, and Asia. Therefore, it contains a combination of swine, avian, and human influenza virus genes. More studies need be conducted to identify the unrecognized molecular markers for the ability of S-OIV A (2009 H1N1) to replicate and be transmitted in humans. As a result these additional studies would help us to determine the mechanism by which an animal influenza A virus crossed the species barrier to infect humans. Additionally, these molecular determinants can be used to predict viral virulence and pathogenicity for diagnosis. 1. LITERATURE REVIEW 1.1. Introduction â€Å"Swine flu† †influenza A [Family Orthomyxoviridae (like influenza B and C viruses), Genus Influenzavirus A] is currently the greatest pandemic disease threat to humankind (Salomon and Webster, 2009). The incidence and spread in humans of the â€Å"swine flu† influenza A virus has raised global concerns regarding its virulence and initially regarding its pandemic potential. The main cause of the â€Å"swine flu† has been identified to be the human infection by influenza A viruses of a new H1N1 (hemagglutinin 1, neuraminidase 1) subtype, or â€Å"2009 H1N1 strain† (Soundararajan et al., 2009) that contains genes closely related to swine influenza (SI) [also called swine flu, hog flu and pig flu]. Thus, the strains of virus that cause the annual seasonal flu are different than the new swine flu viruses that emerged in the spring of 2009. Consequently, as it will be analyzed in the subsequent chapters, the new swine flu virus has a unique combinatio n of gene segments from many different sources (a combination that has not been previously reported among swine or human influenza viruses) and specifically is thought to be a mutation of four known strains of the influenza A virus, subtype H1N1: 1. one endemic in (normally infecting) humans, 2. one endemic in birds, 3. and two endemic in pigs (swine). According to Yoon and Janke (2002), the constant evolution of influenza A viruses through mutation and reassortment present a complex and dynamic picture which is to be unfolded in the remaining Literature Review section more specifically for the H1N1 2009 virus. 1.2. Influenza Influenza is historically an ancient disease of global dimension that causes annual epidemics and, at irregular intervals, pandemics. Influenza is an infection of the respiratory tract caused by the influenza virus (see  § 1.3). When compared with the majority of other viral respiratory infections (such as the common cold), the infection by influenza often causes a more severe illness (Smith, 2003). Influenza-like illness (ILI) is defined by the CDC (Centers for Disease Control and Prevention) as fever (with temperature above 37,8 °C) and either cough or some throat in the absence of any other known cause. According to Webster (1999), influenza is the paradigm of a viral disease in which the continued evolution of the virus is of paramount importance for annual epidemics and occasional pandemics of disease in humans which is attributed to the fact that the H1N1 virus does not fit to the strict definition of a new subtype for which most of the population has not any experience of previous infection (Sullivan et al, 2010) as it is justified later in this Literatute Review section ( § 1.8). Influenza is transmitted by inhalation of microdroplets (because the transmission via large-particle droplets requires close contact which is attributed to the fact that these large-particle droplets cannot remain suspended in the air for a long period of time) of respiratory secretions, often expelled by coughing or sneezing, that contain the virus or from other bodily fluids (such as fomites, diarrheal stool etc.). The incubation period is between 1 to 5 days. Symptoms typically include fever, headache, malaise, myalgia, cough, nasal discharge, and sore throat. In severe cases of influenza, a secondary bacterial pneumonia can lead to the death of a patient (Suguitan and Subbarao, 2007). Vaccination and antiviral treatment constitute the two major options for controlling influenza and are the most effective means of preventing influenza virus infection and further transmission in humans. 1.2.1. Pandemic Influenza An influenza pandemic is a large-scale global outbreak of the disease, whereas an epidemic is considered more sporadic and localized. The aforementioned (in the Summary section) situation of pandemic influenza occurs when a previously circulated human influenza A virus [although all the three types (A, B, and C) of influenza viruses can infect humans)] acquires novel antigenic determinants from an animal-origin influenza virus and now can infect and propagate in humans in the absence of any pre-existing immunity (see  § 1.7 for details). Several influenza subtypes have infected humans. Historical accounts led us to consider that an average of three influenza pandemics have occurred each century, at intervals ranging from 10 to 50 years (Garcia-Sastre, 2005). The three influenza pandemics which occurred in the previous (20th) century are: 1. The â€Å"Spanish† influenza pandemic of 1918 (H1N1 subtype), 2. The 1957 â€Å"Asian flu† (H2N2), and 3. The 1968 ‘‘Hong Kong flu (H3N2). These pandemics resulted in high morbidity, death, and also considerable social and economic disruption. They provide health authorities information on which to base preparations for a future pandemic.The first influenza pandemic of the 21st century, due to a new strain of A(H1N1) virus, was declared on 11 June 2009 by the Director-General of the World Health Organization (WHO) [Collin et al., 2009] by raising the H1N1 flu virus pandemic alert level to phase 6 as it was mentioned in the Summary section. Although influenza B viruses do not cause pandemics, during some epidemic years they have caused more significant mortality and morbidity than influenza A viruses (FLUAV) [Garcia-Sastre, 2005]. 1.3. Influenza Virus It was already mentioned that influenza viruses are divided into three types designated A, B, and C (according to the antigenic differences of their internal structural components as it is discussed below in the current chapter). Influenza types A and B are responsible for epidemics of respiratory illness that occur almost every winter and are often associated with increased rates for hospitalization and death. As it was mentioned in the previous chapter, influenza A virus has also the capability of developing into pandemic virus. Type C infection usually causes either a sporadic mild or asymptomatic respiratory illness or no symptoms at all (Smith, 2003). In comparison to B and C influenza types which are specific to humans, type A viruses can have different hosts, both birds and different mammals (e.g. horses and pigs) including humans (Ã…sjà ¶a and Kruse, 2007). Specifically, influenza B virus strains appear to infect naturally only humans and have caused epidemics every few years (Schmitt and Lamb, 2005). On the other hand, influenza A viruses are significant animal pathogens of poultry, horses and pigs, and multiple antigenically diverse strains exist in a aquatic wild bird reservoir (Garcia-Sastre, 2005). Migrating aquatic birds carry viruses between the continents and thereby play a key role in the continuing process of virus evolution (Murphy et al., 1999). Influenza C virus causes more limited outbreaks in humans and according to Schmitt and Lamb (2005) also infects pigs. Although influenza viruses belong to the best studied viruses, according to Haller et al. (2008), the molecular determinants, which govern the increased virulence of emerging virus strains in humans and which may be associated with their transmission and transmissibility, are presently not well understood. Influenza viruses are negative-strand RNA[1] viruses with a segmented genome (which replicates in the nucleus of the infected cell) belonging to the Orthomyxoviridae family. The morphology of the influenza virion is described in the next chapter. On the basis of antigenic differences influenza viruses are divided into influenza virus types A, B and C. Influenza A viruses are classified on the basis of the antigenic properties of their haemagglutinin (H or HA) and their neuraminidase (N or NA) structural spike-shaped surface glycoproteins (antigens): to date, 16HA (H1-H16) and 9NA (N1-N9) subtypes have been identified (Osterhaus et al., 2008) which gives a theoretical possibility of 144 serological subtypes. Subtypes of influenza A viruses are constantly undergoing small antigenic modifications (antigenic drift) [which is a serotypic change] due to the accumulation of point mutations in their genetic material. In addition, due to the segmented genome, genetic reassortment occurs perio dically when HA and NA genetic material is exchanged between viruses, thereby causing major antigenic changes (antigenic shift) [Yoon and Janke, 2002], the emergence of a new subtype (Smith, 2003) and perhaps the potential for a pandemic outbreak. Both antigenic shift and drift are discussed in  § 1.7. The family Orthomyxoviridae, except the aforementioned influenza viruses A, B and C, also contains the Thogoto viruses. Thogoto viruses are transmitted by ticks and replicate in both ticks and in mammalian species and are not discussed as part of this assignment (Schmitt and Lamb, 2005). 1.4. Influenza Virus Virion This paragraph describes the (belonging to the Orthomyxoviridae family) virus virion[2] morphology. These virions are spherical or pleomorphic, 80-120 nm in diameter (see 1). Some of them have filamentous forms of several micrometers in length. The virion envelope[3] is derived from cell membrane lipids, incorporating variable numbers of virus glycoproteins (1-3) and nonglycosylated proteins (1-2) [Fauquet et al., 2005]. 1. (Left) Diagram of an Influenza A virus (FLUAV) virion in section. The indicated glycoproteins embedded in the lipid membrane are the trimeric hemagglutinin (HA), which predominates, and the tetrameric neuraminidase (NA). The envelope also contains a small number of M2 membrane ion channel proteins. The internal components are the M1 membrane (matrix) protein and the viral ribonucleoprotein (RNP) consisting of RNA segments, associated nucleocapsid protein (NP), and the PA, PB1 and PB2 polymerase proteins. NS2 (NEP), also a virion protein, is not shown (Fauquet et al., 2005). (Right) Negative contrast electron micrograph of particles of FLUAV. The bar represents 100 nm (Fauquet et al., 2005). The lipid envelope is derived from the plasma membrane of the cell in which the virus replicates and is acquired by a budding process (see  § 1.5) from the cell plasma membrane as one of the last steps of virus assembly and growth (Schmitt and Lamb, 2005) which is initiated by an interaction of the viral proteins. Virion surface glycoprotein projections are 10-14 nm in length and 4-6 nm in diameter. The viral nucleocapsid (NP) is segmented, has helical symmetry, and consists of different size classes, 50-150 nm in length (Fauquet et al., 2005). The nucleocapsid segments (the number of which depends on the virus type) surround the virion envelope which has large glycoprotein peplomers (HA, NA, HE). There are two kinds of glycoprotein peplomers[4]: (1) homotrimers of the hemagglutinin protein (NA) and (2) homotetramers of the neuraminidase protein (NA) [see 1 and 2]. Influenza C viruses have only one type of glycoprotein peplomer, consisting of multifunctional hemagglutinin-esterase molecules (HE) [see  § 1.4.1 for further details]. Genomic segments have a loop at one end and consist of a molecule of viral RNA enclosed within a capsid composed of helically arranged nucleoprotein (NP) as it is shown in 2 (Murphy et al., 1999). 2. Schematic representation of an influenza A virion showing the envelope in which three different types of transmembrane proteins are anchored: the hemagglutinin (HA) and the neuraminidase (NA) form the characteristic peplomers and the M2 protein, which is short and not visible by electron microscopy. Inside the envelope there is a layer of M1 protein that surrounds eight ribonucleoprotein (RNP) structures, each of which consists of one RNA segment covered with nucleoprotein (NP) and associated with the three polymerase (P) proteins (Murphy et al., 1999). The aforementioned in the previous paragraph NP protein (arginine-rich protein of approximately 500 amino acids) is the major structural protein of the eight RNPs and it has been found to be associated with the viral RNA segments. Each NP molecule covers approximately 20 nucleotides of the viral RNAs. The NP mediates the transport of the incoming viral RNPs from the cytoplasm into the nucleus by interacting with the cellular karyopherin/importin transport machinery. In addition, the NP plays an important role during viral RNA synthesis, and free NP molecules are required for full-length viral RNA synthesis, but not for viral mRNA transcription (Palese and Garcia-Sastre, 1998). 1.4.1. Influenza Viral Proteins Influenza A and B viruses possess eight single-stranded negative-sense RNA segments (see 2) that encode structural and nonstructural proteins [NS][5]: 1. Hemagglutinin (HA), a structural surface glycoprotein that mediates viral entry (see  § 1.5 for further details) by binding (the HA1 fragment) to sialic acid residues (present on the cell surface) on host fresh target cells, is the main target of the protective humoral immunity responses in the human host (Suguitan and Subbarao, 2007). HA is primarily responsible for the host range of influenza virus and immunity response of hosts to the infection (Consortium for Influenza Study at Shanghai, 2009). After the binding, the virus is taken up into the cell by endocytosis. At this point, the virus is still separated by the endosomal membrane from the replication and translation machinery of the cell cytoplasm (Fass, 2003). HA is initially synthesized and core-glycosylated in the endoplasmic reticulum (ER)[6] as a 75-79 kDa precursor (HA0) which assembles into noncovalently linked homo-trimers. The trimers are rapidly transported to the Golgi complex and reach the plasma membrane, whe re HA insertion initiates the process of assembly and maturation of the newly formed viral particles (33-35). Just prior to or coincident with insertion into the plasma membrane, each trimer subunit is proteolytically and posttranslationally cleaved into two glycoproteins (polypeptides), HA1 and HA2 ( 3), which remain linked by a disulfide bond (Rossignol et al., 2009) and associated with one another to constitute the mature HA spike (a trimer of heterodimers). In that way, the membrane fusion during infection is promoted. Cleavage activates the hemagglutinin (HA), making it ready to attach to receptors on target cells (Murphy et al., 1999). Conclusively and in addition, the HA undergoes various post-translational modifications during its transport to the plasma membrane, including trimerization, glycosylation, disulfide bond formation, palmitoylation, proteolytic cleavage and conformational changes (Palese and Garcia-Sastre, 1998). HA1 is the subunit distal from the virus envelope, whereas HA2 contains a hydrophobic region near the carboxy terminus that anchors the HA1-HA2 complex in the membrane ( 3) [Fass, 2003]. The HA complex is brought to the cell surface via the secretory pathway and incorporated into virions, along with a section of cell membrane, as the virus buds from the cell. HA1 is the subunit distal from the virus envelope, whereas HA2 contains a hydrophobic region near the carboxy terminus that anchors the HA1-HA2 complex in the membrane (see 3) [Fass, 2003]. 3. Primary structure of influenza HA and spatial organization of subunits with respect to the membrane. Cleavage of the influenza HA precursor protein HA0 yields the two subunits HA1 and HA2. HA1 is white, the fusion peptide and transmembrane segments of HA2 are black, and the remainder of HA2 is cross-hatched. For clarity, a monomer of the HA1-HA2 assembly is shown. The amino and carboxy termini of HA2 are labelled ‘‘N and ‘‘C, respectively (Fass, 2003). 2. Neuraminidase (NA) is the other major surface glycoprotein, whose enzymatic function allows the release of newly formed virions, permits the spread of infectious virus from cell to cell, and keeps newly budding virions from aggregating at the host cell surface. This catalytic function of the NA protein is the target of the anti-influenza virus drugs oseltamivir (Tamiflu[7]) and zanamivir (Relenza7). Although these compounds do not directly prevent the infection of healthy cells, they limit the release of infectious progeny viruses thus inhibiting their spread and shortening the duration of the illness. These NA inhibitors are effective against all NA subtypes among the influenza A viruses and may be the primary antiviral drugs in the event of a future pandemic as it proved true in the current â€Å"swine flu† influenza A outbreak. Antibodies to the NA protein do not neutralize infectivity but are protective (Suguitan and Subbarao, 2007). Influenza C viruses lack an NA protein, and all attachment, entry and receptor destroying activities are performed by the aforementioned single spike glycoprotein: hemagglutinin-esterase-fusion (HEF) protein (Garcia-Sastre, 2005). The HEF protein distinguishes the antigenic variants of the genus C of the Orthomyxoviridae family, and the antibody to HEF protein neutralizes infectivity (Schmitt and Lamb, 2005). Of the three virus types, A and B viruses are much more similar to each other in genome organization and protein homology than to C viruses, which suggests that influenza C virus diverged well before the split between A and B viruses (Webster, 1999). Three proteins comprise the viral polymerase of the influenza viruses: two basic proteins (PB1 and PB2) and an acidic protein (PA). They are present at 30 to 60 copies per virion. The RDRP (RNA-dependent RNA polymerase) complex consists of these 3 polymerase proteins (Lamb and Krug, 2001). Together with the aforementioned scaffold protein NP (helically arranged nucleoprotein), these three polymerase proteins associate with the RNA segments to form ribonucleoprotein (RNP) complexes (Murphy et al., 1999). Thus, the RNPs contain four proteins and RNA. Each subunit of NP associates with approximately 20 bases of RNA (Lamb and Krug, 2001). The RNP strands usually exhibit loops at one end and a periodicity of alternating major and minor grooves, suggesting that the structure is formed by a strand that is folded back on itself and then coiled on itself to form a type of twin-stranded helix (Schmitt and Lamb, 2005). RDRP transcribes the genome RNA segments into messenger RNAs (mRNA). The RDR P complex carries out a complex series of reactions including cap binding, endonucleolytic cleavage, RNA synthesis, and polyadenylation[8]. The PA protein may be involved in viral RNA replication and, in addition, the expression of the PA protein in infected cells has been associated with proteolytic activity. The functional significance of the latter activity is not yet understood (Palese and Garcia-Sastre, 1998). Two viral RNA segments (7 and 8) encode at least two proteins each by alternative splicing. Gene segment 7 (see 4) codes for two proteins: matrix protein M1, which is involved in maintaining the structural integrity of the virion, and M2, an integral membrane (surface) protein that acts as an ion channel and facilitates virus uncoating. It is widely believed that the M1 protein interacts with the cytoplasmic tails of the HA, NA, and M2 (or BM2) proteins and also interacts with the ribonucleoprotein (RNP) structures, thereby organizing the process of virus assembly (Schmitt and Lamb, 2005). The drugs amantadine and rimantadine bind to the influenza A M2 protein and interfere with its ability to transport hydrogen ions into the virion, preventing virus uncoating. Amantadine is only effective against influenza A viruses (Suguitsan and Subbarao, 2007). Therefore, for the antiviral therapy, there are two classes of drugs which are currently available for the chemoprophylaxis and the treatment of influenza (Rossignol et al., 2009). These include the aforementioned NA inhibitors oseltamivir and zanamivir, which impair the efficient release of viruses from the infected host cell, and amantadine and rimantadine, which target the viral M2 protein required for virus uncoating. Passively transferred antibodies to M2 can protect animals against influenza viruses, but such M2-specific antibodies are not consistently detected in human convalescent sera (Black et al., 1993), suggesting that this type of immunity may play a minor role in the clearance of influenza virus in humans. Gene segment 8 (see 4) is responsible for the synthesis of the nonstructural protein NS1 and nuclear export protein (NEP, formerly called NS2) [Murphy et al., 1999] which is a minor structural component of the viral core and that mediates nucleo-cytoplasmic trafficking of the viral genome (Garcia-Sastre, 2005). NEP (NS2) plays a role in the export of RNP from the nucleus to the cytoplasm. NS1 protein suppresses the antiviral mechanism in host cells upon viral infection (Chang et al., 2009) and is involved in modulating the hosts interferon response (Garcia-Sastre, 2005). Recently, an unusual 87-amino acid peptide arising from an alternative reading frame of the PB1 RNA segment has been described (Chen et al., 2001). This protein, PB1-F2, is believed to function in the induction of apoptosis[9] as a means of down-regulating the host immune response to influenza infection. Specifically, it appears to kill host immune cells following influenza virus infection. It has been called the influenza death protein (Chen et al., 2001). PB1 segment encodes this second protein from the +1 reading frame. This protein consists of 87-90 amino acids (depending on the virus strain). This protein is absent in some animal, particularly swine, virus isolates. PB1-F2 protein is not present in all human influenza viruses. Human H1N1 viruses encode a truncated version. However, it is consistently present in viruses known to be of increased virulence in humans, including the viruses that caused the 1918, 1957, and 1968 pandemics. PB1-F2 localizes to mitochondria and treatment of cells with a synthetic PB1-F2 peptide induces apoptosis9 (Neumann et al., 2008). 4. Orthomyxovirus genome organization. The genomic organization and ORFs are shown for genes that encode multiple proteins. Segments encoding the polymerase, hemagglutinin, and nucleoprotein genes are not depicted as each encodes a single protein. (A) Influenza A virus segment 8 showing NS1 and NS2 (NEP) mRNAs and their coding regions. NS1 and NS2 (NEP) share 10 amino-terminal residues, including the initiating methionine. The open reading frame (ORF)[10] of NS2 (NEP) mRNA (nt 529-861) differs from that of NS1. (B) Influenza A virus segment 7 showing M1 and M2 mRNAs and their coding regions. M1 and M2 share 9 amino-terminal residues, including the initiating methionine; however, the ORF of M2 mRNA (nt 740-1004) differs from that of M1. A peptide that could be translated from mRNA has not been found in vivo. (C) Influenza A virus PB1 segment ORFs10. Initiation of PB1 translation is thought to be relatively inefficient based on Kozaks rule[11], likely allowing initiation of PB1-F2 translation by ribosomal scanning (Fauquet et al., 2005). In the same way, the M2 protein is anchored in the viral envelope of the influenza A virus, the ion channel proteins BM2 (it is encoded by a second open reading frame10 of RNA segment 7 of influenza B virus, and its function has not been determined) and CM2 are contained in influenza B and C viruses respectively ( 5). The CM2 protein is most likely generated by cleavage of the precursor protein. The influenza B viruses encode one more transmembrane protein, or NB, of unknown function (Garcia-Sastre, 2005). The cellular receptor for the influenza C virus is known to be the 9-0-acetyl-N-acetylneuraminic acid, and its receptor-destroying enzyme is not an NA, as it was already mentioned, but a neuraminate-O-acetylesterase. Like the HA protein of A and B viruses, the HEF of influenza C viruses must be cleaved in order to exhibit membrane fusion activity (Palese and Garcia-Sastre, 1998). 1.5. Viral Entry Influenza virus infection is spread from cell to cell and from host to host in the form of infectious particles that are assembled and released from infected cells. A series of events must occur for the production of an infectious influenza virus particle, including the organization and concentration of viral proteins at selected sites on the cell plasma membrane, recruitment of a full complement of eight RNP segments to the assembly sites, and the budding and release of particles by membrane fission (Schmitt and Lamb, 2005). Viral entry is a multistep process that follows at ­tachment of the virion to the cellular receptor and re ­sults in deposition of the viral genome (nucleocapsid) in the cytosol[12] (receptor-mediated endocytosis). The entry of enveloped viruses is exemplified by the influenza virus ( 6). The sequential steps in entry include (Nathanson, 2002):  § Attachment of the HA spike [the virus attachment protein (VAP)] to sialic acid receptors (bound to glycoproteins or glycolipids) on the cellu ­lar surface (see  § 1.4.1 for further details). This step contributes to pathogenesis, transmission, and host range restriction.  § Internalization of the virion into an endocytic vacuole.  § Fusion of the endocytic vacuole with a lysosome[13], with marked lowering of the pH (see 6). In endosomes, the low pH-dependent fusion occurs between viral and cell membranes. For influenza viruses, fusion (and infectivity) depends on the cleaved virion HA (FLUAV and FLUBV: HA1, HA2; FLUCV: HEF1, HEF2) [Murphy et al, 1999]. The infectivity and fusion activity are acquired by the post-translational cleavage of the HA of the influenza viruses which is accomplished by cellular proteases. Cleavability depends, among other factors, on the number of basic amino acids at the cleavage site. It produces a hydrophobic amino terminal HA2 molecule (Fauquet et al., 2005). 6. Diagram of the stepwise entry of influenza virus at a cellular level. Key events are attachment of the virion; internalization of the virion by endocytosis; lowering the pH of the endocytic vacuole leading to drastic reconfiguration of the viral attachment protein (hemagglutinin, HA1 and HA2); insertion of a hydrophobic domain of HA2 into the vacuolar membrane; fusion of the viral and vacuolar membranes; release of the viral nu ­cleocapsid into the cytosol (Nathanson, 2002).  § A drastic alteration in the structure of the HA1 trimer, with reorientation of the HA2 peptide to insert its proximal hydrophobic domain into the vacuolar membrane (Nathanson, 2002).  § Fusion of viral and vacuolar membranes (Nathanson, 2002).  § Integral membrane proteins migrate through the Golgi apparatus to localized regions of the plasma membrane (Fauquet et al., 2005).  § New virions form by budding, thereby incorporating matrix protein and the viral nucleocapsids which align below regions of the plasma membrane containing viral envelope proteins. Budding is from the apical surface in polarized cells (Fauquet et al., 2005).  § Release of the viral nucleocapsid into the cy ­tosol: After the formation of fusion pores, viral ribonucleoprotein complexes (RNPs) are delivered into the cytosol. RNPs are then transported into the nucleus, where transcription and replication occurs (see 7) [Garten and Klenk, 2008]. How the replication and the transcription of the genome of influenza virus take place in the nuclei of infected cells is summarized in detail by Palese and Garcia-Sastre (1998) [ 7]. (1) Adsorption: the virus interacts with sialic acid-containing cell receptors via its HA protein, and is intenalized by endosomes. (2) Fusion and uncoating: the HA undergoes a conformational change mediated by the acid environment of the endosome, which leads to the fusion of viral and cellular membranes. The inside of the virus also gets acidified due to proton trafficking through the M2 Ion channel. This acidification is responsible for the separation of the M1 protein from the ribonucleoproteins (RNPs), which are then transported into the nucleus of the host cell thanks to a nuclear localization Signal in the NP. (3) Transcription and replication: the viral RNA (vRNA) is transcribed and replicated in the nucleus by the viral polymerase. Two different species of RNA are synthesized from the vRNA template: (a) full-length copies (cRNA), which are used by the polymerase to produce more vRNA molecules; and (b) mRNA. (4) Translation: following export into the cytoplasm the mRNAs are translated to form viral proteins. The membrane proteins (HA, NA and M2) are transported via the rough endoplasmic reticulum (ER) and Golgi apparatus to the plasma membrane. The viral proteins possessing nuclear signals (PB1, PB2, PA, NP, M1, NS1 and NEP) are transported into the nucleus. (5) Packaging and budding: the newly synthesized NEP protein appears to facilitate the transport of the RNPs from the nucleus into the cytoplasm by bridging the RNPs with the nuclear export machinery. M1-RNP complexes are formed which interact with viral proteins in the plasma membrane. Newly made viruses bud from the host cell membrane (Palese and Garcia-Sastre, 1998). 1.5.1. Sialic Acid Receptors of Influenza Viruses Sialic acids (Sias) are a family of negatively charged 9-carbon sugars typically occ